For sequence determination of DNA having long base sequence such as a human genome, a DNA sequencer having high sensitivity, a high speed and large throughput is necessary. As one method thereof, a multi-capillary DNA sequencer arranging capillary columns charged with gels in plural is proposed in place of that employing flat plate type slab gels. In the capillary column, a sample is not only easy to handle or inject but also can be migrated at a high speed and detected in high sensitivity as compared with the slab gel. Namely, such problems result from influence by Joulean heat that a band spreads and a temperature gradient takes place applying a high voltage in the slab gel, while such problems are less in the capillary column and spreading of a band is small to allow high sensitivity detection even if making high-speed migration while applying a high voltage.
When making electrophoresis employing a plurality of capillary columns, it is desirable that attachment/detachment of the capillary columns to/from an electrophoretic apparatus body is easy, and it is desirable to cassette the same with a holder for that. In consideration of the recent environmental problem, it is also preferable that the capillary columns can be readily separated from the holder when discarded after use.
Furthermore, since a step by pressurization or decompression is requisite for charging gels into the capillary columns, airtightness is necessary for fixation of the capillary columns to the holder at least on sample injection sides in order to simultaneously charge the gels into all capillary columns contained in the cassette.
Accordingly, a first objective of the present invention is to provide a capillary cassette which can readily separate capillary columns from a holder and has sufficient airtightness between the capillary columns and the holder on sample injection sides.
The capillary columns are so thin that the same are difficult to manually work and are easily breakable. When fixing the capillary columns to the holder, it is a difficult operation requiring skill to bundle the same in plural, and in addition, it requires a long time.
Accordingly, a second objective of the present invention is to make it possible to simply and quickly perform preparation of a capillary cassette.
The capillary columns are protected with various coats, in order to increase mechanical strength. For coats, polyimide, silicone resin, polytetrafluoroethylene, acrylic resin and the like are used. When using the same as coats for capillary columns of electrophoresis however, optical characteristics of the coats such as light absorption and fluorescence hinder detection since optical means for absorbance measurement and fluorescence measurement are employed for detection of samples migrating in the capillary columns. Therefore, when the coats are made of materials hindering optical detection, the coats of detection parts are burned by flames and removed with burning means such as a lighter or a gas burner.
A multi-capillary electrophoretic apparatus using a plurality of capillary columns and simultaneously detecting a plurality of samples aligns, arranges and fixes such capillary columns that coats of detection parts are removed, thereby forming detection windows when the coats are made of materials hindering optical detection.
While a number of capillary columns are arranged in the multi-capillary electrophoretic apparatus, it follows that when performing coat removal of the detection parts on every one of the capillary columns, a long time is required for the operation.
Furthermore, the coating materials include such a one that a part located in a flame is completely burned and removed in the process of removing the coating material by burning by the flame while the same is melted but does not come to be removed in a portion separate from the flame and convex parts resulting from solidification of the melted coat are formed on both sides of the part from which the coat is removed. If such convex portions are formed, adjacent capillary columns do not adhere to each other or come to different levels due to the convex portions and detection windows are not planarly aligned when arranging the capillary columns, aligning positions of the parts from which the coats are removed and forming the detection windows. Consequently, the focus of an optical system may so deviate in detection that it cannot perform proper detection.
Accordingly, a third objective of the present invention is, when a coat for a capillary column is made of a material hindering optical detection, to provide such a capillary cassette that a detection window of a capillary column employed for a multi-capillary electrophoretic apparatus is planarly formed to be suitable for optical measurement and a method of manufacturing a capillary cassette having a feature in a detection window forming step thereof.
An initial capillary electrophoretic apparatus is that employing a single-capillary column. In this case, it dips an end of the capillary column into a gel solution in a vessel, closes the vessel and pressurizes the vessel thereby pushing the gel into the capillary column and charging the same as a method of charging the gel into the capillary column. As another method, an operation of dipping an end of the capillary column into the gel solution, decompressing another end of the capillary column and sucking the gel into the capillary column, thereby charging the same, is also performed.
In multi-capillary electrophoresis, it is necessary to align capillary column end surfaces on sample injection sides in sample injection while it is necessary to align and handle capillary coat removing parts on detection sides when performing detection for handling a plurality of capillary columns, and hence it is desirable to provide such a capillary cassette that arrangement is fixed by a holder so that both the sample injection sides and the detection sides are in prescribed arrangement relation.
Furthermore, it is extremely troublesome to charge a gel into every one of the capillary columns in the state of the capillary cassette, and, in actuality, impossible in practice. Therefore, awaited is means for making it possible to simply perform gel charging into all capillary columns included therein in units of the capillary cassette.
Accordingly, a fourth objective of the present invention is to provide an apparatus to make it possible to simply charge gels into all capillary columns included in such a capillary cassette.